Numerous agricultural and industrial production systems and processes depend on specific organisms, such as plants, algae, bacteria, fungi, yeasts, protozoa and cultured animal cells, for production of useful materials and compounds, such as food, fiber, structural materials, fuel, chemicals, pharmaceuticals, or feedstocks. In the process of the current shift to biological production systems for a variety of chemicals and fuels, a wide assortment of organisms will be used for production, most of them microbes, with an increasing tendency towards photosynthetic organisms (Dismukes 2008). The ability to grow robustly, and the ability to efficiently produce the materials and compounds of interest, are desirable properties of these organisms.
Optimization of the growth of these organisms and augmentation of their yield of useful materials and compounds is an ongoing activity of many companies and individuals, with the goal of achieving a higher productivity or yield, or lower production cost of commercially important materials and compounds. Such improvements can occur through the modification of production systems, or through the modification of the organisms themselves.
Polynucleotide fusions, involving joining of intact or partial open reading frames encoded by separate polynucleotides, is a known way of altering a polynucleotide sequence to change the properties of the encoded RNA or protein and to alter the phenotype of an organism. There are two general mechanisms by which polynucleotide fusions can alter an organism's phenotype. These two mechanisms can be illustrated with the case of polynucleotide A (encoding protein A′) fused to polynucleotide B (encoding protein B′), in which proteins A′ and B′ have different functions or activities and/or are localized to different parts of the cell. The first mechanism applies to sub-cellular localization of the two proteins. The fusion protein encoded by the polynucleotide fusion of the two polynucleotides may be localized to the part of the cell where protein A′ normally resides, or to the part of the cell where protein B′ normally resides, or to both. This alteration of cellular distribution of the activities encoded by proteins A′ and B′ may cause a phenotypic change in the organism.
The second general mechanism by which fusion proteins alter the phenotypic property of a cell or organism relates to the direct association of two different, normally separate functions or activities in the same protein. In the case of proteins A′ and B′, their fusion may lead to an altered activity of protein A′ or of protein B′ or of the multiprotein complex in which these proteins normally reside, or of combinations thereof. The altered activity includes but is not limited to: qualitative alterations in activity; altered levels of activity; altered specificities of activity; altered regulation of the activity by the cell; altered association of the protein with other proteins, DNA or RNA molecules in the cell, leading to changes in the cell's biochemical or genetic pathways. As a result, a system for creating artificial polynucleotide fusions has the potential to create many phenotypes that are rarely or never found in nature.
To date, no attempt has been made to take advantage of the function-generating capability of fusion polypeptides in a large-scale and systematic manner. There are no published examples of large-scale collections of randomized, in-frame polynucleotide fusions. Previous examples of fusion proteins have been generated in a limited and directed fashion with specific outcomes in mind. The present invention describes the creation and use of systematic, randomized, large-scale and in-frame polynucleotide fusions for the purpose of altering protein function, generating new protein functions, and/or generating novel phenotypes of interest in biological organisms.
The present invention also describes methods by which large-scale collections of randomized, in-frame fusion polynucleotides can be selected in an iterative fashion to arrive at smaller collections of in-frame fusion polynucleotides enriched for a particular function or ability to confer a phenotype of interest to an organism.